1. Field of the Invention
The present invention describes a new enzyme-catalyzed synthetic process for the production of 1-acyl-2-lyso and 1,2-di-acylated glycerophospholipids and their synthetic or natural analogues, wherein the acyl groups in the mono- and di-acyl compounds which may be the same or different each derived from a saturated or unsaturated, short-, medium- and long-chained linear or branched free carboxylic acid or a derivative thereof, preferably a free fatty acid or a derivative thereof, selected from the group consisting of fatty acid chloride, fatty acid alkyl ester, fatty acid vinyl ester, fatty acid anhydride and any other activated form of a fatty acid serving as a fatty acyl donor. In accordance with the present invention, there is provided an enzymatic esterification/transesterification (acylation) process for the production of 1,2-diacylated and 1-acylated-2-lyso phospholipids using as substrate glycerophosphorylcholine (GPC), or analogue derivative thereof, where the choline moiety can be substituted by ethanolamine, serine, inositol, glycerol or any other alcohol, together with a fatty acid derivative, as defined above. The reaction can be performed in a solvent or in a solvent-free microaqueous system, in the presence of a phospholipase which may be immobilized onto an insoluble matrix and is optionally surfactant coated (modified). The process of the present invention leads to the formation of 1-acyl-2-lyso-glycerophospholipids and 1,2-di-acylated glycerophospholipids, with a high conversion rate.
The present invention relates to the development of an enzymatic process for preparing 1-acylated- or 1,2-di-acylated phospholipids, and their synthetic or natural analogues. More specifically, it relates to a process for preparing 1,2-diacyl-3-glycerophospholipids and 2-lyso-3-glycerophospholipids of the formulae STR and STR1 respectively, wherein R and R′ are the same or different and are each derived from a saturated or unsaturated, short-, medium- and long-chained linear or branched free carboxylic acid or derivative thereof, preferably a free fatty acid or derivative thereof, selected from the group consisting fatty acid chloride, fatty acid alkyl ester, fatty acid vinyl ester, fatty acid anhydride and any other activated form of a fatty acid serving as a fatty acyl donor. X in both formulae represents choline, serine, ethanolamine, glycerol, inositol, or any other appropriate alcohol moiety. 
2. Prior Art
Phospholipids are the main structural components of the cell membrane. Soybeans and egg yolk are the major natural sources for obtaining phospholipids in commerce. This class of materials has been well recognized for their surface-active properties, therefore, phospholipids find extensive use in the food, cosmetics and pharmaceutical industries. The type of fatty acyl residues at the sn-1 and sn-2 positions (sites) in natural phospholipids vary, and their proportion in general depends on their source. For some practical applications it is favored to have phospholipids of defined structure with respect to the type of fatty acyl bound to the sn-1 or sn-2 positions of the glycerol skeleton. In some applications, other carboxylic acyl groups are needed, which may be derivatives of either fatty- or non-fatty acids. Phospholipids of defined structure are usually obtained from natural sources by fractionation or by means of liquid chromatography. Different chemical synthesis approaches have also been developed for the production of phospholipids with specific fatty acyl groups. The most widely practiced method for obtaining 1,2-diacylated phospholipids is based on a non-enzymatic reaction in which GPC or other glycerophospholipids react with an activated fatty acid derivative such as fatty acyl chloride or fatty acid anhydride. For example, dimyristoyl, di-stearoyl and di-oleoyl derivatives of phospholipids have been prepared with yields of 51.0%, 38.4% and 54.7%, respectively, by reacting cadmium chloride salt of GPC with the appropriate fatty acid chloride at room temperature for 3 hours (U.S. Pat. No. 4,130,571). When the reagents and the products of this reaction are in contact with each other for a prolonged time the percent of the formed by-products increased significantly. Furthermore, when using the fatty acyl chloride method, the preparation of phospholipids having highly unsaturated fatty acyl groups was not efficient for producing the desired products (U.S. Pat. No. 4,130,571). The chemical, non-enzymatic, synthesis route developed for the production of 1,2-diacylated phospholipids starting from GPC and fatty acid anhydride has shown improved results and less by-products (U.S. Pat. No. 4,130,571). For example, according to this method, di-palmitoyl derivative (yield: 90%), di-stearoyl derivative (yield: 81%) and di-oleoyl derivative (yield: 71%) were obtained by reacting cadmium chloride salt of GPC with the appropriate fatty acid anhydride and tetraethyl ammonium salt of the fatty acid. The preparation of polyunsaturated fatty acid derivatives in this process has encountered the production of high percentage of cyclic by-products, and consequently, the yield for the production of this type of phospholipids was relatively low (U.S. Pat. No. 4,130,571). In other words, the 1,2-diacyl yield is high when the fatty acid used as a substrate is saturated or monounsaturated, while it is low when the fatty acid is polyunsaturated.
Furthermore, the chemical synthesis of 1,2-diacyl-phospholipids, which utilizes a variety of acidic or basic reagents and environments, may harm the chiral center of natural phospholipids, a fact that is of crucial importance in pharmaceutical as well as in other applications.
A recent promising synthetic method to replace existing acyl groups in phospholipids with desired ones, has been developed based on using natural phospholipids as starting materials. Special enzymes have been used to perform this type of reaction in organic media. Many recent reports indicate (Adlercreutz P. et al., J. Mol. Cat B: Enzymatic 11, p. 173-178 (2001) and Aura A. M. et al. JAOCS, Vol 72, no. 11, p. 1375-1378 (1995)) that the fatty acyl moiety on the sn-1 and sn-2 positions in phospholipids can be replaced using different types of hydrolases, such as specific or non-specific lipases with broad substrate specificity and phospholipase A2. Basically, many reports have shown that different lipases derived from various species are capable of incorporating specific fatty acids on the sn-1 position of phospholipids. For example, U.S. Pat. No. 6,268,187 describes an esterification process for preparing a lysophospholipid using lipase in the presence of glycerol-3-phosphate derivative, a fatty acid derivative and one or more salt hydrate pairs. Carmen Virto and Patrick Adlercreutz, Enzyme and Microbial Technology Vol. 26, 630-635 (2000), have demonstrated that immobilized lipase from Candida antarctica lipase B (Novozyme 435) was effective in the synthesis of lysophosphatidylcholine. The transesterification of glycerophosphorylcholine and vinyl laurate was carried out in a solvent-free system or in the presence of 50% (v/v) t-butanol. The lipase was selective for the sn-1 position of the glycerol backbone and almost no phosphatidylcholine was produced in the first 24 hours of the reaction. However, and probably due to acyl migration, the formation of phosphatidylcholine increased slowly if the reaction was incubated over a long period of time. Incorporation of a specific fatty acid on the sn-2 position using phospholipase A2, was demonstrated. However, so far no practical and efficient method has been reported. Pernas, T. et al., Biochemical and Biophysical Research Communications, Vol. 168(2), 644-650 (1990), demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine.
WO 91/00918 teaches a method for the preparation of a phospholipid with a carboxylic acid residue in the 2-position wherein a lysophospholipid is esterified with a corresponding carboxylic acid in the presence of the catalyst phospholipase A2, the esterification taking place in a microemulsion with a water content of 0.1-2% by weight. According to this publication a 1,2-diacyl-glycerophospholipid (for example, 1,2-diacyl-PC) is formed from 2-lyso-glycerophospholipid (2-lyso-PC) in the presence of a carboxylic acid and phospholipase A2. The reported yields of obtained 1,2-diacylated-phospholipids are in the range of 7-12%.
It seems that the highest yield for obtaining phospholipids having the desired acyl group on the sn-2 position using phospholipase A2 for catalyzing the interesterification of 1,2-diacylphospholipid and a specific, desired fatty acid derivative, was reported to reach only the range of 6-7% (Svensson, I. JAOCS, Vol 69, No. 10, p. 986-991 (1992)). These reactions have basically been performed in a microaqueous organic medium or in a bi-phase reaction medium. The main disadvantage of using these methods for the production of tailor-made phospholipids is the competing hydrolysis reaction at the sn-2 site to yield 1-acyl-2-lyso-phosphatidyl choline as a by-product, which significantly reduces the recovery of the desired 1,2-diacyl-phospholipids. In addition, the degree of incorporation of a specific fatty acid at a desired position on the glycerol backbone of the phospholipid, using different combinations of enzymes, is generally low, and in most cases did not exceed 20% (Aura A. M. et al. JAOCS, Vol 72, no. 11, p. 1375-1378 (1995)). Although many lipases and phospholipases derived from various sources of microorganisms appear in the literature as involved in phospholipid modification, none of these enzyme preparations has been found to catalyze the production of 1,2-diacylated phospholipids having a desired structure in high yields.
An alternative method has recently been developed for the production of phospholipids with desired fatty acids on the sn-1 and sn-2 positions (U.S. Pat. No. 5,654,290). This method is based on a two-step enzymatic-chemical method wherein the first step constitutes the production of 2-lyso-phosphatidylcholine starting from glycerophosphorylcholine (GPC), a defined fatty acid derivative and an appropriate enzyme in microaqueous system. Lipases, such as Rhizomucor miehei lipase and Novozym 435 (Virto et al., Enzyme and Microbial. Technol., 26, p. 630-635, 2000)) are in general capable of catalyzing this type of reaction and to give relatively high yields of the product. The second step of the reaction is carried out by mixing the purified 1-defined acyl-2-lyso-phosphatidylcholine with an activated fatty acyl donor, such as fatty acid anhydride or fatty acyl chloride, in an organic solvent in the presence of a chemical catalyst to obtain the appropriate 1,2-diacyl-phosphatidylcholine. The enzymatic synthesis of lysophosphatidylcholine with lipases has been demonstrated in microaqueous organic media using GPC and different fatty acid derivatives as starting materials (Virto et al., Enzyme and Microbial. Technol. 26, p. 630-635 (2000)). Conversions of 70-88% of GPC to yield 1-acyl-2-lyso-phosphatid choline were achieved with different fatty acid derivatives, whereas up to 12% of 1,2-diacyl-phosphatidylcholine was formed as a by-product in the reaction. In most of these studies, the enzyme lipase B of Candida antarctica (Novozym 435) has been used for specific acylation at the sn-1 position of the glycerophospholipid and to produce the 1-acylated phospholipids. Due to migration of the 1-acyl group to the sn-2 position a consecutive interesterification reaction on the sn-1 hydroxyl group leads to the formation of 1,2-diacylphospholipids as a by-product (Virto et al., Enzyme and Microbial. Technol. 26, p. 630-635 (2000)). The obtained results in these reported studies showed that the lipase was very selective for the sn-1 position and the formation of 1,2-diacyl-phospholipids was not detected during the first hours of the reaction. Furthermore, only after most of the substrate GPC was consumed, the slow formation of 1,2-acyl-phosphatidylcholine was observed. In these studies, 1,2-diacyl-phosphatidylcholine was most likely formed by the migration of the acyl from the sn-1 position to the sn-2 position. It was indicated that high yields of 2-lysophosphatidylcholine were achieved in short time when vinyl ester of fatty acid or fatty acid anhydrides were used due to the favorable thermodynamic equilibrium. Furthermore, a high excess of fatty acid vinyl ester to GPC is necessary to achieve significant conversions.
Neither of the above discussed publications has demonstrated an enzymatic esterification/transesterification process for preparing a substantial yield of 1,2-diacyl-glycerophospholipid nor they demonstrated an esterification/transesterification process, using phospholipase A1, for preparing 2-lyso glycerophospholipid from glycerophospholipid.
The preparation of 1,2-diacylated phospholipids in a highly efficient one-step process which should provide substantial economic benefits. Consequently, it is an object of the present invention to provide an efficient, enzymatic process for preparing 1,2-pre-determined, identical diacyl-phospholipid, preferably 1,2-diacyl-PC comprising the use of an enzyme preparation, referred to as PLA1,2, in microaqueous reaction systems. It is yet a further object of the present invention to provide the said 1,2-pre-determined identical diacyl-phospholipid in a one-step enzymatic process. It is yet an additional object of the present invention to use the said PLA1,2 enzyme preparation or a phospholipase enzyme that catalyzes acylation at the sn-1 position (site), referred to as PLA1, for converting glycerophospholipid, preferably GPC, into 1-acyl-2-lyso-glycerophospholipid, preferably 1-acyl-2-lyso-PC.